DOI: 10.

1177/0003702820920652

Paper type: Special Issue: Microplastics Submitted Paper

Microplastics Differ Between Indoor and Outdoor Air Masses:
Insights from Multiple Microscopy Methodologies
Emily Gaston,1,* Mary Woo,1 Clare Steele,1 Suja Sukumaran,2 Sean Anderson3
1
California State University Channel Islands, Environmental Science and Resource Management
Program, CA 93012 USA
2
Thermo Fisher Scientific, San Jose, CA 95134 USA
3
California State University Channel Islands, Environmental Science and Resource Management
Program, PIRatE Lab, CA, 93012 USA
*Corresponding author email: Emily.Gaston@csuci.edu

Abstract
The abundance and distribution of microplastic (<5 mm) has become a growing concern,
particularly over the past decade. Research to date has focused on water, soil, and organism
matrices but generally disregarded air. We explored airborne microplastic inside and outside of
buildings in coastal California by filtering known volumes of air through glass fiber filters,
which were then subsequently characterized with a variety of microscopy techniques: gross
traditional microscopy, fluorescent microscopy following staining with Nile red, micro-Raman
spectroscopy, and micro-Fourier transform infrared (FT-IR) spectroscopy. Microplastics
permeated the air, with indoor (3.3±2.9 fibers and 12.6±8.0 fragments m–3; mean±1 SD)
harboring twice as much as outdoor air (0.6 ±0.6 fibers and 5.6±3.2 fragments m–3). Microplastic
fiber length did not differ significantly between indoor and outdoor air, but indoor microplastic
fragments (58.6±55 µm) were half the size of outdoor fragments (104.8±64.9 µm). Micro-Raman
and FT-IR painted slightly different pictures of airborne plastic compounds, with micro-Raman
suggesting polyvinyl chloride dominates indoor air, followed by polyethylene (PE) and micro-
FT-IR showing polystyrene dominates followed by PE and polyethylene terephthalate. The
ubiquity of airborne microplastic points to significant new potential sources of plastic inputs to
terrestrial and marine ecosystems and raises significant concerns about inhalation exposure to
humans both indoors and out.
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Keywords: Pollution, polymer, waste, inhalation, ecotoxicology, air quality, microspectroscopy,

Raman, Nile Red, Fourier transform infrared spectroscopy, FT-IR

Introduction
Microplastic (<5 mm) has become a ubiquitous environmental contaminant worldwide1 since the
start of large-scale industrial plastic production during and in the immediate wake of World War
II.2 Annual plastic production reached 233.75 million metric tons (MT) in 2013 and is expected
to grow to 334.83 million MT per annum by 2020.3 Of the 1.3 billion MT of waste generated in
urban centers globally in 2010, approximately 21% (275 million MT) was plastic.4 Most of this
plastic waste was managed in energy recovery or landfills, but an estimated 31.9 million MT
were classified as mismanaged and therefore a potential source of plastic pollution to the wider
environment. An estimated 4.8 to 12.7 million MT of plastic waste entered the ocean in 2010.4
Early reports of plastic litter in the ocean emerged in the 1970s,5 with researchers encountering
plastic items spanning a variety of sizes from micrometers to meters. Researchers have
investigated the abundance of plastic in marine and freshwater environments and the potential
effects on associated biota with increasing interest over the ensuing decades, with nearly half of
the 17100 citations for “microplastic” emerging since 2014 according to Google Scholar. The
predominant focus on aquatic systems has shown microplastic present from remote areas such as
polar regions6 and the deep sea7–9 to heavily populated urban river catchments,10,11 estuaries,12,13
and beaches.14 Recently, researchers have begun searching farther afield and documenting
microplastic in varied systems including floodplain soils,16 sewage sludge-amended soils,16,17
subalpine lake sediments in Lake Garda, Italy,18 and rainwater falling on Paris, France.19
Discovery of microplastic across so many ecosystems suggests this now-ubiquitous pollutant is
yet another marker of the Anthropocene.20
Methodological discrepancies exist throughout the marine and freshwater microplastic
literature when it comes to identifying microplastic. Research groups have identified
microplastic using various combinations of the following methodologies: tactile response, hot
wire melt tests, visual quantification, fluorescence after Nile red staining, and spectroscopic
methods of µRaman and/ or µFT-IR. These discrepancies largely stem from accessibility, cost,
and rapid pace of change within the research community. Given this lack of standardized
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characterization methods, we set out to evaluate several approaches in a way that could provide
guidance to future microplastic researchers.
Recent studies examining microplastic abundance in France’s urban Paris21 and rural
Pyrenees mountains22 concluded that microplastic can be transported through the air and
deposited into terrestrial and aquatic environments.18 Atmospheric deposition is understood as
the flux of substances from the atmosphere onto the earth’s surface wherein dry and wet
deposition combine to produce total atmospheric fallout.23 Microplastic fallout may prove to be a
heretofore underappreciated, major upstream source of microplastic into both terrestrial and
aquatic environments. As such, a thorough understanding of the density and distribution of
microplastic in air is critical to estimating their potential environmental and human health risks.23
Airborne microplastic is a potential threat to humans and terrestrial vertebrates when inhaled,
respired, and potentially retained within lungs.19,23–25 Microplastics smaller than 25 µm can enter
the human body through the nose or mouth, and those less than 5 µm can end up in lung tissue.26
Regulatory concern around fine particulates in the air has grown significantly in recent years
(e.g., California Air Resources Board’s Particulate Matter Program),27 particularly around the
smallest sized anthropogenic fragments linked to a wide range of health impacts including
asthma28 and heart attacks.29 Most countries have air pollution standards to limit the volumes of
fragments less than 10 and 2.5 µm, collectively known as PM 10 and PM 2.5 standards.26
Interior airspaces within built environments are significant (e.g., the City of Los Angeles alone
contains more than 1.7 billion cubic meters of officially recognized structures27) and air within
such structures could prove to be a microplastic source or sink for the wider environment. The
volume of microplastic entering our environment is likely to increase given likely increases in
plastic production worldwide.30
Three recent studies in France and Germany investigated the density and distribution of
microplastic in urban and rural outdoor air.19,22,23 These studies demonstrated larger fragments
present in atmospheric deposition and suggested that local wind-blown debris played a major
role in adding to the number of fragments found in bulk samples. Harrison et al.31 found airborne
fragments outside tend to have an upper size limit of 100 μm, whereas fragments in indoor dust
are smaller with maximum dimensions around 10 μm.
In this study, we specifically asked (i) whether microplastic loads differ between indoor
and outdoor air masses, and (ii) if four different spectroscopic methods produced consistent
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conclusions. If different methodologies yield different conclusions, some of the recently

published work exploring microplastic pollution could be in question.
This research provides evidence of the occurrence of airborne microplastic at California
State University Channel Islands (CSUCI), a semi-urbanized landscape in coastal California. We
quantified and compared microplastic density (plastic fibers and fragments m–3) between indoor
and outdoor air masses, explored different polymer composition within indoor and outdoor air
with µRaman and micro-attenuated total reflection Fourier transform infrared microscopy
(µATR FT-IR), and suggest some best practices for use of these technologies for the burgeoning
field of microplastic characterization.

Methods
Microplastic Nomenclature
Most microplastic we have encountered in various settings in the past three years can be readily
classified into one of three gross morphological categories: microspheres (or microbeads,
machined spheres with constant diameter designed as abrasives in retail products or industrial
processes), fibers (filamentous), and fragments (irregular shaped pieces of plastic often ovoid or
trapezoidal). Some research groups distinguish additional fourth category of nurdles (pre-
production plastic pellets used as the feedstock for commercial manufacturing); however, in
practice we cannot consistently distinguish weathered nurdles from fragments. While we have
isolated each of our three common microplastic morphologies from air, this particular data set
presented herein captured only fibers and fragments, so our comments will be restricted to these
two categories of microplastic.

Air Monitoring Efforts

As part of a larger monitoring effort, we sampled a wide array of air masses across 100 km of
coastal Southern California from January 2019 through the end of March 2019. The bulk of this
data is being used to characterize human inhalation exposure over the greater coastal zone. For
purposes of this paper, we report on a subset of samples collected across the campus of CSU
Channel Islands (CSUCI) to compare four microplastic characterization methods; gross visual
quantification, fluorescence after Nile red staining, µFT-IR spectroscopy, and µRaman
spectroscopy.
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Sample Locations
Air masses across the CSUCI campus are likely representative of the wider California coastal
airshed spanning Santa Barbara and Ventura Counties. CSUCI lies within a wider, semi-
urbanized landscape matrix harboring many major industrial, agriculture, and commercial
activities nested between densely populated cities and large swaths of natural
landscapes/protected areas in coastal southern California. The 337 ha main campus is 8 km
inland and sits on the extreme western edge of the Santa Monica Mountains where they intersect
with the Oxnard Plain in Ventura County, home to intense row crop, orchard, and greenhouse
agriculture. Other major activities within 10 km of campus include large biotechnology,
semiconductor, and aerospace clusters, a major military base, a large general aviation airport,
and the 101 Freeway corridor (~200000 vehicle trips per day).29 CSUCI was formally opened in
2002 within the former Camarillo State Mental Hospital campus whose buildings date from the
1930s. As such, occupied University structures are now a diverse matrix of buildings spanning
construction dates from 1932 to 2017 with a corresponding mixture of heating, ventilation, and
air conditioning systems and environmental envelopes creating a range from interior air masses
treated with state of the art air handling and purification systems to spaces with minimal air
handling/segregation between external and interior air.
We report data from air sampled air at five locations throughout the CSUCI campus
(Table I) between January 2019 and March 2019. At four of these locations, paired air samples
were collected to assess microplastics density inside and outside of respective buildings. Two to
three replicates were collected at each sampling site, constrained by the logistics of rotating a
single sampling apparatus and field researchers’ availability. Each air sample was collected
during active work week periods (between Monday and Friday) when students were in class
session (i.e., excluding breaks or weekends). Each of these sampling sites were within 3–6 m of a
building entrance. Outdoor air only was sampled at our fifth site (Student Union Quad),
approximately 10 m from the nearest building entrance.
We collected a total of 21 ambient air samples (10 indoor, 11 outdoor) plus their paired
procedural controls (n=21) for various levels of analyses: gross traditional microscopy, Nile red
stain with fluorescence microscopy, and/or microspectroscopy (µFT-IR or µRaman). While all
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samples were inspected with gross microscopy and Nile red fluorescence, the distinct sample
preparation for µFT-IR and µRaman translated into any given replicate being characterized by
either FT-IR or Raman (not both).

Quality Assurance and Quality Control

All measures for microplastic contamination were employed consistently throughout our
laboratory and field efforts. First, all researchers handling samples or equipment wore natural
fiber clothing at all times in the field and laboratory, with cotton lab coats donned for all sample
processing and analysis. Glassware including Petri dishes, vacuum filtration funnels, and
tweezers for handling filters, were triple rinsed with doubly filtered deionized (DFDI) water
immediately before use. All of our DFDI water utilized for rinsing and sample processing in this
study was twice filtered through 1.6 µm filters to remove ambient fragments and plastics present
in traditional research-grade deionized water generation systems, thereby reducing sample
secondary contamination. Our double filtration yielded water with ≤3.4 microplastic fibers or
fragments per liter (3.4±0.4, n=40 L; mean±1 SD). Filters were kept covered at all times after
initial sampling, except during imaging and spectrographic characterization steps. Finally,
procedural controls were run alongside each sample to quantify contamination from equipment
and reagents in samples.

Sample Collection
We sampled airborne microplastic with a simple vacuum filtration array (Fig. 1) that drew
ambient air through a glass microfiber filter (GF/A or GF filter; Whatman #1820-047; nominal
pore size of 1.6 µm; 47 mm diameter) secured in a vacuum filter holder with a Cole-Parmer Air
Admiral vacuum pump (model # P-79202-00). Air flow averaged 11.7 L min–1 for a minimum of
eight hours, continuously collecting microplastic and other particulates on the GF filter.
Extensive preliminary testing showed an eight-hour sample duration guaranteed a sufficient
particulate load for analysis. The amount of air pumped through the system was tracked with an
airflow totalizer (Alicat Scientific mode # M-100SLPM-D15M) so fragment count could be
expressed per volume of air sampled. An average of 4615 L were filtered over the eight-hour
sampling windows. Each site was sampled two to three times, with a procedural control included
during each sampling event. This procedural control consisted of a second vacuum filter holder
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with GF filter and vacuum flask, but lacked a pump and added a lid over the filter holder. This
covering eliminated any ambient microplastic fallout from the atmosphere. This procedural
control served to quantify secondary contamination from equipment, reagents in samples, etc.
and allowed us to define our limit of detection (LOD)/quantification (LOQ) for microplastic. Our
pumps were plugged into standard electrical outlets to provide a constant voltage, avoiding the
sometimes-inconsistent voltage and therefore pump rates that can result from powering such a
system with external battery power.

Figure 1. Vacuum filtration setup with filter, filter holder, GF filter (top left), vacuum flask
(bottom left), airflow totalizer (middle), and vacuum pump (right). The procedural control was
identical to the fist elements, but not connected to a pump and with a cap added over the filter
folder. This procedural control was placed adjacent to the sampling array.
Sample Processing
Any microplastics that settled around the inside of the filter holder were rinsed down onto the GF
filter with DFDI water at the end of each given round of sampling. We then removed the GF
filter from its filter holder, placed it into a cleaned glass petri dish, and immediately covered it.
The fibers and fragments collected on GF filters were not put through a digestion procedure to
remove organic matter (e.g., cellulose) as we routinely do when analyzing microplastic from
other matrices and environments. The fiber and fragment densities on our filters were low
enough that we could easily individually visualize and categorize any suspected fragment as
plastic, mineral, or other organic matter.
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Quantification with Traditional Microscopy

In our laboratory, we photographed each GF filter with a stereoscope (Olympus, model SZ61)
integrated with a digital camera (Lumenera Infinity 2) under 40x magnification. We then visually
quantified particulates embedded on the filter surface after Hidalgo-Ruz,5 categorizing each by
morphology (fiber or fragment) and color. Enumeration followed a consistent, systematic search
grid pattern to provide a complete census of the entire filter surface. We scored fragments and
fibers as apparent microplastic if they were bright in color, reflective, sharp, fraying or had a
length five times their width (standard fiber). Our visual limit of detection (i.e., the smallest fiber
or fragment consistently identifiable) was 20 µm.

Quantification with Microscopy and Nile Red

Nile red can improve quantification of microplastics because it is a lipid soluble fluorescent dye
that stains hydrophobic materials.32,33 Following our visual quantification, we exposed our filters
to 0.5–1 mL of 10 µg/mL of Nile red solution (made up as 1 mg/mL in acetone and diluted in n-
hexane).34 Filters were allowed to dry with the upper petri dish lid slightly askew in the fume
hood for a minimum of 10 min. We again quantified apparent microplastic on these stained
filters under a 450–510 nm LP fluorescent light (Orion-Lite 455 nm #OL-455NG) for excitation
and an orange 529 nm filter for viewing subsequent fluorescence.32 Fibers and fragments were
scored as microplastic if they fluoresced under these conditions.

Microplastic Morphometrics
All fibers and fragments upon 20 randomly selected filters (six indoor air samples, ten outdoor
air samples, two indoor procedural controls, and two outdoor procedural controls) were
measured with an NIH Image Fiji.35 We measured the total length of each fiber and maximum
width for each fragment. While NIH Image can batch process large numbers of targets via
automated routines, the diversity of target morphologies drove us to conduct all measurements
manually. Length and width data were log transformed to better meet assumptions of normality
and then compared with one-way analysis of variance (ANOVA) with SPSS (IBM v.24.0.0.0)
software.

Spectrographic Characterization
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We further characterized a subset of the fibers and fragments found on the GF filters by micro-
Raman (µRaman) and micro-Fourier transform infrared (µFT-IR) spectroscopy. Both of these
spectrographic methods have been applied to characterize fibers and fragments isolated from
different matrices and confirm the presence of microplastic.22,36,37 Furthermore, these two
methods are complementary,38–40 with each yielding somewhat different information about the
fibers and fragments isolated from air in this study.

µRaman Spectroscopy
We generated spectra of Raman shifts in the 200–2000 range with a Horiba Scientific Xplora
Plus confocal Raman microscope (Raman shift: 50–3200 cm, 1.5 cm–1 resolution, imaging
accuracy 0.5 µm) with a motorized x,y,z stage. Our general settings consisted of a 785 nm laser
at 25–100% power (filter), 1200 mm–1 gratings with a 100 µm split, and 10–15 s acquisition
times (i.e., fiber and fragment exposure to the laser) and five to 10 accumulations (averaged to
generate a final spectrum). We adjusted our default settings to maximize signal strength and
minimize fluorescence and damage to the fibers and fragments being examined. Two other laser
wavelengths, 532 nm and 683 nm, were available and spectra were attempted with them.
However, with the sample presentation used here and in the majority of analyses conducted, our
strongest signal and minimum fluorescence were observed with the 785 nm laser. Higher laser
power (i.e., 50% and 100%) and the higher energy lasers (i.e., 532 nm and 683 nm) were more
likely to burn the fiber and fragment being examined.22,41 Spectrum from any fiber or fragment
burned during analysis were not utilized and the item was re-analyzed in a different area if
possible. There were, however, a number of fibers that withstood the 785 nm laser at 100% with
no visible damage.
We selected, at random, 155 fibers and fragments from five indoor and six outdoor filters
for individual Raman analysis, generating 23 usable spectra for indoor samples and 44 usable
spectra for outdoor samples. We had less usable spectra from Raman analysis compared to FT-
IR because of high fluorescence and burning of fibers and fragments. Four procedural control
filters were similarly examined under our Raman confocal microscope, with a Raman spectrum
generated for each fiber or fragment encountered. In addition, two GF filters stained only with
Nile red (no other processing), were examined in the same manner (termed Nile red blanks).
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Raman spectra were analyzed through the Bio-Rad ExpertID software (18.3.111.0) with
KnowItAll spectral databases AnalyzeIt Raman (18.3.111.0). The baseline and noise correction
factors available via ExpertID were applied to process raw spectra prior to library searches.
Composite spectra (two to four component) with a library-sample-match of ≥ 90% were utilized.

µFT-IR Spectroscopy
Our µFT-IR analysis used a Thermo Scientific Nicolet iN10Mx microscope. Because glass fibers
yield a strong signal IR signal, fibers and fragments could not be analyzed on the original GF
filters used for collection. Instead, fibers and fragments were removed from the GF filters and
spread out on a glass slide with a stream of DFDI water. The fibers and fragments were allowed
to completely dry and then individually analyzed with a germanium micro-attenuated total
reflection (ATR) interface on the FT-IR microscope with a mercury–cadmium–telluride (MCT)
detector. The smooth background of the slide did not interfere with micro-ATR analyses,
whereas the 3D textured glass fibers pose issue with reflection measurements. Spectra were
collected at a resolution of 8 cm–1 and 64 scans were coded to yield a final absorbance spectrum
from 650–4000 cm–1.
The FT-IR spectra baselines were corrected, and the spectra analyzed through
ThermoFisher OMNIC Picta software (v.1.7) populated with extensive polymer spectral
databases. Spectra yielding a single component library-sample-match of >90% are reported here.
We selected four indoor and four outdoor air filters spanning our sampling locations for FT-IR
analysis. From these filters we characterized 121 fibers and fragments via FT-IR, generating 59
usable spectra from indoor and 62 from outdoor samples.

Reported Microplastic Density

Unless stipulated as “raw” microplastic counts, all reported microplastic density data herein are
adjusted to reflect potential contamination. For blank correction, we subtracted the relevant (i.e.,
indoor fiber, indoor fragment, outdoor fiber, or outdoor fragment) contaminant load present on a
paired procedural control from the raw microplastic counts measured upon our experimental
filters, yielding the corrected microplastic loads. Further, all fiber and fragment counts on each
filter were divided by the volume of air pumped through the filter, as recorded by the airflow
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totalizer in standard liters, multiplied by a 10³ conversion factor and reported as fibers or
fragments per m³.

Results
Background Contamination
Our procedural controls had median values of 8.1 fibers and 4.7 fragments present on the indoor
sample filters (n=10) and 0.6 fibers and 7.3 fragments per outdoor sample (n=11) when assessed
with gross microscopy. These procedural controls had an aggregate (fibers + fragments)
microplastic contamination of 12.8 ± 4.0 (mean ± 1 SD) across all indoor filters and 12.0 ± 3.4
across all outdoor filters, translating into a contamination load of less than one microplastic item
per 2.9 cm² of filter area. Our Nile red procedural controls had median values of 2.4 fibers and
12.2 fragments per indoor filter (n=10) and 0.4 fibers and 6.3 fragments per outdoor filter
(n=11). Aggregate (fibers + fragments) Nile red controls harbored 5.3±5.1 microplastic items per
filter.
We used the above fiber and fragment values to correct our raw microplastic densities out
of an abundance of caution. However, we were only able to confirm a single polymer (a red
fragment from an outdoor control) upon our procedural control filters with Raman and FT-IR
spectroscopy. All other items (n=13) successfully characterized from our control filters were
mineral, carbon, or some other non-plastic material. In short, our contamination control
procedures for polymer contamination were effective and our corrections provide a conservative
overall estimate of microplastic abundance.

Fiber and Fragment Sizes

Fiber lengths did not differ significantly (F1,301=2.017, p=0.157) between indoor (641±810.7 µm,
mean ± 1SD) and outdoor (616±536.7 µm) air masses, although our largest fibers were found
indoors (Table II). Maximum indoor fiber length (8961 µm) was more than four times longer
than the largest outdoor fiber (2061 µm) encountered. Our most abundant fiber length category
was 101–301 µm for both indoor (26.6±0.1% of all indoor fibers) and outdoor (29.7±0.1% of all
outdoor fibers) air masses (Fig. 2).
In contrast, fragments were significantly (F1,280=46.866, p<0.001) smaller in inside
buildings. Outdoor (104.8±64.9 µm) microplastic fragments were nearly twice as large as their
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indoor (58.6±55 µm) counterparts on average (Table II). As with all of the fiber distributions we
observed, outdoor fragments were most abundant at intermediary size ranges with 39.2±0.1% of
fragments in the 76–100 µm range (Fig. 2). Indoor fragments were strongly dominated by the
smallest size classes with 59.2±0.1% in our smallest size range (<50 µm) and followed a strongly
exponential distribution.

Figure 2. Size distribution for all fibers (top) and fragments (bottom) counted and measured on
sample GF filters. Bins are 25 µm for fragments and 200 µm for fibers (due to the greater range
of sizes). Bars: mean ± 1SD.

Traditional Microscopy: Fibers

Interior air spaces were significantly enriched with microfiber relative to outdoor spaces
(F =13.757, p=0.001; Fig. 3, Table III). Gross visual microscopy counts showed indoor air
1,20

contained many more fibers (9.8±7.3 fibers per m³; mean±1 SD) than outdoor (1.5±1.1 fibers per
m³). Indoor sites segregated into relatively fiber-dense and fiber-sparse locations. The Broome
Library foyer (17.0±3.8 fibers per m³) and Sierra Hall main entrance (15.4±3.1 fibers per m³)
were fiber-rich spaces and our Modoc (3.0 ±0.6 fibers per m³) and ESRM (3.2 ±0.4 fibers per
m³) laboratories were comparatively fiber-poor.
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Outdoor microfiber abundance was comparable to our low-fiber interior spaces, but all
outside fiber concentrations were below the levels of all sampled interior spaces. Outdoor fibers
fluctuated little between our facilities parking lot (adjacent to our Modoc laboratory; 2.2±01.8
fibers per m³), campus mall (1.7±0.7 fibers per m³), ESRM patio (1.3±0.2 fibers per m³), Student
Union plaza (1.1±0.7 fibers per m³), and Broome fountain (0.7±0.1, fibers per m³).

Traditional Microscopy: Fragments

Interior air spaces had significantly fewer microplastic fragments relative to outdoor spaces
(F1,20=12.500, p=0.002; Fig. 3, Table III). In aggregate, interior spaces had roughly half (6.7±5.2
fragments per m³) the fragments of outdoor (15.5±6.1 fragments per m³) regions. In descending
rank, interior fragment densities were; Broome Library foyer (8.3±6.3 fragments per m³), ESRM
laboratory (7.9±5.6 fragments per m³), Sierra Hall foyer (6.7±6.8 fragments per m³), and Modoc
laboratory (2.7±2.7 fragments per m³).
Outdoor microplastic fragment abundance ranged from highs of 19.6±2.0 (Broome
fountain) and 18.1±7.9 (Facilities parking lot) to 13.2±5.6 (ESRM patio), 12.9±2.4 (campus
Mall), and 12.5±10.6 (Student Union plaza) fragments per m³.

Traditional Microscopy: Apparent Diversity

The apparent diversity of aggregated fibers and fragments was greater in interior spaces (Fig. 3),
primarily due to more evenly distributed categories. Black fragments strongly dominated outdoor
air microplastics, accounting for 45% of the total outdoor items observed.
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Figure 3. Microplastic fiber (top) and fragment (middle) counts per m3 of air sampled across
CSUCI from traditional microscopy. Bars: mean ± 1 SD. Abundance of the total number of
microplastics (bottom) fibers (solid) and fragments (dotted) counted across all sample GF filters
binned by color (left to right: black, blue, red, green).

Nile Red Fluorescence

Samples processed with Nile red mirrored the trends already described between indoor and
outdoor microfiber, but with only approximately one-third of the microplastic counts observed
with gross microscopy (Table III). Indoor air again harbored significantly (F1,20=31.358,
p<0.001) more microfiber (3.3±2.9 fibers per m³; mean±1 SD) than outdoor (0.6±0.6 fibers per
m³; Fig. 5). For microplastic fragments however, the indoor-outdoor pattern inverted itself such
that interior spaces (12.6±8.0 fragments per m³) now showed twice the fragment abundance as
outdoor (5.6±3.2 fragments per m³) spaces, a significant difference (F1,20=13.055, p=0.004). As
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with our microfibers, absolute fragment counts were also approximately one-third of the
estimates from gross microscopy for our outdoor samples. The increase in fragments counted
upon our indoor filters with Nile red was driven by many exceedingly small items (generally <10
µm). These exceedingly small fragments stained/fluoresced strongly and were therefore much
easier to detect and count compared to our bright field approach used with the previous gross
microscopy technique. In short, our enumeration ability greatly improved with these bright
objects on a dark background (e.g., Fig. 4).

Figure 4. Examples of Nile red stained microplastics under ambient light (left) and blue light
(455 nm) viewed with an orange filter (529 nm; right)
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Figure 5. Microplastic fiber (top) and fragment (bottom) density per m³ of air sampled across
CSUCI from microscopy with Nile red. Bars: mean ± 1 SD.

Chemical Composition Spectroscopy: µFT-IR

Our 122 fibers and fragments successfully characterized with µFT-IR spectroscopy (Fig. 6) were
dominated by non-plastic compounds (90%, n=110), including inorganic substances (46%, n=56)
and cellulose (45%, n=54; Fig. 7b, Table III). Polystyrene (PS) dominated (46%, n=5) aggregate
µFT-IR plastic polymer signals with polyethylene terephthalate (PET; 36.4%, n=4), polyethylene
(PE; 9%, n=1), and acrylic (9%, n=1) also encountered. Micro-FT-IR painted a rather
homogeneous landscape of plastics both indoors and out. Indoor plastic polymers consisted of PS
(n=4) and PET (n=3). We encountered only singleton spectra from polymers (PET, PS, and
acrylic) on outdoor filters (Fig. 7d, Table III).

Chemical Composition Spectroscopy: µRaman

Of the 71 fibers and fragments we successfully characterized with µRaman spectroscopy (Fig.
6), 38 (54%) were synthetic plastic polymers. The remainder were fingerprinted as non-polymer
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anthropogenic substances (15%, n=11), mineral or cellulose (24%, n=17), and dyes or simply
unknown (7%, n=5; Fig. 7a, Table III). Polyvinyl chloride heat stabilizer (PVC-HS) was the
most common plastic detected overall with µRaman, comprising 50% (n=19) of our identified
plastics and dominating both indoor (n=7) and outdoor (n=12) plastic signals. Polymetric plastic
additives (polymeric) comprised 24% (n=9) of our aggregate plastic items. All other plastics
were detected only once or twice: polyvinyl chloride (PVC; 5%, n=2), polyethylene (PE; 5%,
n=2), resin (5%, n=2), acrylic (6%, n=2), polycarbonate (PC; 3%, n=1), and polystyrene (PS;
3%, n=1; Fig. 7d, Table III).
Our various air masses presented generally similar plastic compounds as characterized by
µRaman. Indoor plastic polymers primarily consisted of PVC-HS 47% (n=7), polymeric 40%
(n=6), and PE 13% (n=2). Outdoor plastic polymers were similar, consisting of 57% (n=12)
PVC-HS, 10% (n=2) PS, and 10% (n=2) polymeric.

Plastic Identifiers
Our spectrographic characterizations did not always rely on a “pure” polymer signal itself. We
frequently utilized spectra of additives associated with a given commercial formulation of a
plastic item’s identity. Our most common plastic identifiers were PVC additives (52%, n=19),
Dibutyl-dichlorotin (79% n=15) and Valfor 100 Zeolite (21%, n=4), both common heat
stabilizers in PVC.37 Another common spectrum (n=4) was the polymeric additive
polyphosphoric acid (PPA) used to improve the properties of polymer-modified asphalts.42 The
polymeric additive 1,2,3 triazole (n=4) is a photostabilizer in polymers with strong antimicrobial
and antifouling properties integrated into many plastics but most commonly into polystyrene.43
We also encountered the copper phthalocyanine (n=1) dye used in various polymers.37 The
remaining plastic spectra were common “pure” plastic polymer signals of PVC (n=2), PE (n=2),
Resin (n=2), Acrylic (n=1), PC (n=1), and PS (n=1).
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Figure 6. Representative images and spectra from µFT-IR (top two panels) and µRaman (bottom
two panels).
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Figure 7. Abundance of different materials detected using µRaman (a) and µFT-IR (b), and
relative abundance of the different polymeric materials detected using µRaman (c) and µFT-IR
(d) in subsampled fragments and fibers. PVC=polyvinyl chloride, PVC-HS=polyvinyl chloride-
heat stabilizer, PE=polyethylene, PS=polystyrene, PC=polycarbonate, PA=polyamide,
ABS=acrylonitrile butadiene styrene, PET=polyethylene terephthalate.

Discussion
Amounts of Microplastics in Air
Visual Quantification. Our results confirm the ubiquitous presence of microplastic in indoor and
outdoor air environments demonstrated in previous studies.13,19,21–23,44–46 Note these previous
studies have sampled both microplastics suspended in air (via vacuum filtration) and
microplastic fallout from air (via bulk deposition samples). This present study focused on the
former (suspended microplastic) as a potential direct exposure pathway to humans. Suspended
indoor air microplastic densities on the CSUCI campus from traditional microscopy, 9.8±7.3
fibers per m3 and 6.8±5.2 fragments per m3, are comparable to the two previous studies: 5.4
fibers per m3 (median) and 9.3±5.8 fragments+fibers per m3.13,21 CSUCI outdoor air suspended
microplastic densities, 1.5±1.1 fibers per m3 and 15.5±6.1 fragments per m3 are consistent with
one previous study, 0.9 fibers per m3 (median).21 The only other study published to date on
suspended outdoor microplastic densities reported inconsistently high values of 1274±390 total
microplastic (fragments+fibers) per m3.46 This final estimate is three orders higher than anything
we have sampled in this or any of our other airborne microplastic monitoring across the United
States (unpublished data), lacked controls, and lacked verification that the substances that were
collected were indeed polymers.
We measured significantly elevated concentration of fibers inside (F1,20=13.757,
p=0.001), with 6.5 times greater density (quite similar to the 5.5x estimate from Nile red
estimates), on average, inside compared to outside air. Dris and coworkers21 reported a similar
trend with fiber densities in indoor (6x greater than outdoor) air. Indoors, clothes made of
synthetic materials, furniture, carpets and air filters are all potential sources of fibers to air and
exist at a higher concentration than outside. Further, inside air has less of a chance of mixing
with fresher/cleaner air and reduced dilution propensity compared to turbid outdoor air masses.
Traditional microscopy showed suspended fragment densities were 2.3x greater outdoors than
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indoors. The majority of our fragments found both indoor and outdoor were black, but many
were not identifiable with either Raman or FT-IR spectroscopy (either retuning no useful spectra
or manifesting as “black carbon”). Intriguingly, at least some of these small black fragments
could ultimately prove to be trace amounts of tire according to a recent study published by the
San Francisco Estuary Institute wherein they state, “nearly half of the fragments from field
[water and sediment] samples were black fragments. Spectroscopic analysis and secondary
characteristics suggested these fragments might be synthetic or natural rubber”.47

Nile Red Quantification

Our microplastic density estimates from Nile red counts varied in comparison to densities from
traditional microscopy. The same filters used during visual quantification were stained with Nile
red and subsequently counted under a blue light with an orange filter. Both indoor and outdoor
fiber and outdoor fragment densities decreased by a factor of 2.5–3.0 with Nile red counts (Table
III). This decrease demonstrates the selectivity of the hydrophobic Nile red that stains synthetic
fibers and fragments and selected organic matter, but not inorganic or hydrophilic materials.
Previous studies with Nile red staining of microplastics have reported that cotton cellulose, wool,
paper products, “black carbon”, algae, and other OH-rich carbohydrate-based polymers only
stain weakly.32,48 Other biogenic materials, such as chitin, wood lignin, and natural waxes can
stain strongly and may lead to false positive counts when such compounds are abundant.32,49
Considering that 42–47% of the fibers examined under µFT-IR were cellulose (Table III) and
would likely not stain (or stain weakly), a decrease in fiber counts is consistent with our
understanding of and experience with Nile red methodology and efficacy. Outdoor fragments
likely comprise a variety of plant matter fragments, some of which will stain, so more data is
needed to investigate the observed trend in terms of selectivity. Further, the decrease in Nile red
counts may also demonstrate the selectivity of Nile red to different polymer types. For example,
PP, PE, and PS stain strongly, but PC, PET, PVC and weathered PE stain to a lesser extent.49
PVC and PET were strong components of the polymeric makeup in µRaman and µFT-IR
analyses (Fig. 7), respectively, and may have only weakly stained; they thereby may well have
been undercounted in our Nile red enumeration.
We saw a markedly distinct pattern with indoor fragments and Nile red, which was the
exact opposite of the trends for fibers and outdoor fragments. Nile red counts for indoor
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fragments almost doubled compared to traditional microscopy. One possibility for the increase is
the staining of chitin material; however, this outcome would be a better explanation in outdoor
air where fragments of arthropod exoskeletons and similar biogenic materials are presumably
more abundant. A second, more likely possibility is that Nile red improves the accuracy of
smaller, clear, and white fragment counts. Beyond the ability to selectively stain plastics, we
have found Nile red improves counts of smaller-sized fragments by creating a clearer fluorescing
signal on a dark background that is more easily visible and counted, something observed by other
researchers as well.32,34,49 Maes et al.32 predict that fragments down to 5 µm may potentially be
visualized, depending upon the optics of the microscope in use. Further, Nile red will illuminate
clear and white fragments that are obscured by the GF (or any other light colored) filter
background. In our study, indoor air appears to have a concentration of small fragments that
were stained and more accurately counted under fluorescing conditions. The possibility that
these fragments are of biogenic origin, rather than polymeric, cannot be ruled out, as no digestion
step was carried out. Nevertheless, the large quantity of small fragments quantified with Nile red
is consistent with the indoor air fragment size distributions (Fig. 2) where small fragments <50
µm comprise 59.2±0.1% of our overall sample pool.

Spatial Variation of Microplastics in Air

Microplastic fiber densities varied between the five sampled sites on the CSUCI campus, while
the fragment densities stayed generally consistent between sites (Fig. 3). The most notable trend
is higher fiber densities encountered at our Sierra Hall foyer and Broome Library sites, and lower
densities at our Modoc, ESRM and Union sites. The Sierra and Broome sites connect well-used
areas of the campus, are major throughways for students and staff, and thus sustains high foot
traffic. Fibers from the synthetic clothing of students and staff passing through could easily
contribute to microfiber loading of the inside air. With estimates of 1900 to 250 x 103 fibers per
garment being released during a single wash cycle50,51 of synthetic clothing, fibers being broken
and shedding as a person crosses a throughway is quite reasonable and a likely source of fibers at
the Sierra and Broome sites. In-progress work by our research group is currently accumulating
evidence that supports this interpretation of fiber shed in high-traffic or densely populated
interior spaces.
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Size of Microplastics in Air

Fragments were more abundant than fibers in both indoor and outdoor air. Of the 668 individual
microplastic targets measured on sample filters, 366 were fragments and 302 were fibers. This is
consistent with previous studies that reported microplastic fragments as the major components in
atmospheric fallout.22,23 Also similar to previous work, fiber and fragment distributions show
highest abundance within smaller size classes and reduced abundance of larger size categories
more easily seen with lower magnifications and more readily manipulated with traditional tools.
Indoor air fragments sizes appear to be following a trend different from outdoor fragments and
all fibers, indoor and out.
The accuracy of fiber and fragment counts decreases approaching our (size) limit of
detection (20 µm). Our data indicate a larger pool of smaller fragments (moving toward
nanoplastics), consistent with other work,21–23 that we likely poorly (if at all) quantified here.
These are an important size class, as these smallest fragments (<5 µm) are the most inhalable and
respirable in terrestrial mammal lungs.52
We observed a number of notable differences in microplastic size distributions between
outdoor and indoor air. First, the size range of fibers and fragments in outdoor air (fiber 25-2061,
fragment 51–408 µm) were smaller than for items in indoor air (fiber 22–8961 µm, fragments
20–850 µm), although overall fiber lengths did not differ significantly (p=0.157) between indoor
and outdoor areas. It is possible that indoor fibers and fragments undergo less weathering (less
exposure to UV sunlight, wind, temperature shifts, etc.) and are thereby somewhat less likely to
fracture than those in outdoor air, potentially explaining the maximum interior fiber size being
more than four times the length of correspondingly outdoor fibers. Further, the stable, low-wind
indoor conditions inside buildings will allow these larger fibers and fragments to more easily
settle out than in the turbid outdoor environment. Dris et al.21 reported a similar trend with fibers
(50–1650 µm for outdoor air and 50–3250 µm for indoor air) but their maximum fiber lengths
were more than half the size of this study. Dris et al.21 sampled air masses at 1.2 m, an average
breathing height, while our study pumped air only 30 cm off the ground. Larger fibers with
greater mass and surface area may lack sufficient buoyancy to reach the 1.2 m breathing height,
and might settle more rapidly or more readily gather on surfaces,22 including this study’s GF
filter resting at just 30 cm off the ground. As always, the different sampling locations (private
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European apartments versus North American university campus) may also influence these
observed differences in fiber length.
Second, and most notably, <50 µm fragments dominate our indoor air (59%), while 50–
100 µm fragments are more abundant in outdoor air (62%). As recognized with our Nile red
analyses, small fragments appear to be concentrating within indoor air. Indoor air has
concentrated sources of fragments (skin cells, tracked-in dirt, fragments from paper goods and
plastic packaging, etc.) that are not as easily diluted as outdoor air. Further, the central HVAC
systems in most of the university buildings sampled employed Merv10 filter (or better) rated to
remove >85% of 3–10 µm fragments and 50–65% of 1–3 µm fragments. As air is cycled through
the HVAC system, some smaller fragments may not be removed, settle as dust and resuspend, or
be cycled back through the HVAC, thereby concentrating in indoor air.

Composition of Microplastics in Air

Our chemical characterization of sampled fibers and fragments with parallel µRaman and µFT-
IR show differences in the polymeric makeup between indoor and outdoor air (Fig. 7). The fibers
and fragments evaluated during µFT-IR analysis were 15% polymeric outdoor and 5% indoor;
the parallel µRaman analysis values were 63% polymeric indoor and 47% outdoor (Fig. 7). The
consistent trend between these methods was the greater polymeric abundance in indoor air
particulates. The remaining content was natural (e.g., cellulose and wool), inorganic (e.g.,
minerals), or simply unidentified materials (Fig. 7).
The relative contributions of polymeric and cellulosic material indoors shown with FT-IR
data (Fig. 7) is lower than the two previous indoor air studies that reported 33% and 50%
polymeric material21 and 67% and 50% natural fiber.14 The dominant polymer also differs
between studies. In this study, FT-IR data was dominated by PS, followed by PE and
PET. Vianello et al.13 reported PE as the dominant polymer in their samples, while Dris et al.19
found PP most abundant. Further, the Raman data from this study shows PVC as most prevalent,
followed by PE.
Outdoor air also had an abundance of natural materials with lower contributions from
polymeric materials (43% cellulose, 5% polymeric; FT-IR, Fig.7). FT-IR data showed an equal
distribution of PS, acrylic and PET polymers, yet Raman data was dominated again by PVC
signals (Fig. 7). Kaya et al.46 reported that PE and polyamides were most abundant in their
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outdoor air samples. No other studies, to our knowledge, have chemically characterized
suspended microplastics in outdoor air. Studies that have examined microplastic fallout in
outdoor air reported PE, ethylene vinyl acetate (EVAS), PET, PE, PP and PS as the major
polymers identified using FT-IR,23,44 and PS, PE, and PP using Raman.22
Many factors influenced these results from sample location to sample processing and
terminal analysis. Terminal analysis, which can be defined the end analysis for further
understanding of combined analyses. For example, Vianello et al.13 and Dris et al.20 sampled in
private apartments and rooftops in large urban European cities, whereas our study evaluated a
semi-urban university campus surrounded by multiple land uses (urban cities, agriculture,
protected areas, etc.). It is reasonable to expect the sources of plastic, and thus the atmospheric
loading, to vary between these quite different sampling locations. Next, each FT-IR analysis was
carried out with different modes (reflectance versus transmission) and on different substrates
(glass slide versus zinc selenide window), which can bias signal strength from different size,
shape and type of polymer. Studies used different terminal analyses: FT-IR and Raman. FT-IR
and Raman signals are often complementary, and it has been documented that PE shows a strong
signal in FT-IR and, oppositely, PVC yields a strong signal in Raman.38,39
Overall, our data show that the distribution of polymer type varies between indoor and
outdoor air. The FT-IR and Raman data together indicate that PE and PET polymer abundance is
greater inside, while PVC and acrylic are found more outside. PE and PET are common in food
packaging (plastic bags and water bottles, to-go containers) and construction materials (vapor
barriers, window films, flooring protection), but most notably as textiles (synthetic clothing,
fleece sweaters and blankets).51 PVC fibers are widely used in outdoor furniture and coverings
due to their ease of use and durability. Non-fibrous PVC has many other applications, but the
PVC microplastics identified here were mainly fibrous.

Insights from Multiple Microscopy Methods

Each of the four microscopy methods applied to microplastic fibers and fragments sampled from
air on the CSUCI campus provide different insights into microplastic sources, pathways and
composition. Traditional microscopy allows gross counts of microplastic densities and
assessment of microplastic morphology (shape, size distribution, etc.), but misidentification can
lead to large errors.53 Nile red staining with microscopy provides selectivity of plastics and a
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lower (size) limit of detection that improves upon traditional microscopy, although it is not a
perfect “plastic only” stain. Raman and FT-IR work together to chemically fingerprint sampled
fibers and fragments. Chemical fingerprints allow for source tracking and investigations of
microplastic pathways into the environment. While FT-IR can identify many polymer types and
natural and inorganic materials (cellulose, wool, silicates), generating a sufficient signal from
small diameter fibers or fragments (<20 µm) is limited and detecting trace components (<1%)
requires complex sample preparation. Raman, in addition to identifying many polymer types, can
be extremely sensitive to trace components, such as dyes and additives, that can further help
fingerprint and allow upstream or downstream source tracking of sampled fiber and fragment
populations. However, in some cases, the dye and additive fingerprints can overwhelm the
polymeric signal. For example, Raman analysis of an acrylic paint chip in Kappler et al.39
revealed a titanium oxide colorant not visible with IR, but required parallel FT-IR analysis to
pinpoint the underlying acrylic polymer. In this study, FT-IR provided a strong fingerprint of
major components comprising subsampled fibers and fragments (cellulose, inorganic, PS, PE,
PET, acrylic), while Raman gave a more detailed fingerprint of the dyes and additives associated
with polymeric and synthetic materials (Fig. 7 and Table III). Dyes and additives in synthetic
material can pose a health risk54 and the level of detail provided by Raman analysis as to their
presence may be important for risk assessments of human exposure to microplastic.

Recommendations for Future Studies

As expected, a post hoc review of the four microscopy methods employed in this study reveal a
number of potential improvements for future studies. Because the field of microplastic
quantification and characterization is in rapid development and no standardized methods have
been globally adopted, it is important to practically share such lessons learned with the greater
scientific community. In this way, the field can move together to prudently compile robust
standardized methods. With such a sentiment in mind, we share our lessons learned during this
study and share resulting recommendations in detail below.

Sample Presentation
When compiling a study design for microplastic evaluations in any environment, it is important
to choose the terminal analysis ahead of time. The four microscopy methods applied in this
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study, traditional microscopy, Nile red staining and microscopy, and characterization via µFT-IR
or µRaman, as well as chemical characterization via pyrolysis–gas chromatography–mass
spectrometry (GC-MS), are increasingly common terminal analyses.55,56 The ideal sample
presentation varies between these terminal analyses and the study design should consider
methods that minimize sample processing and yield the optimal sample presentation for their
chosen terminal analysis.
The initial terminal analysis chosen for this study was traditional microscopy, for which
the use of GF filters was appropriate. The GF filters were low-cost, clean, easy to use (strong,
not brittle and do not tear easily) and provided a clean white background for easy visualization of
collected microplastic. One disadvantage is that white and clear plastics are less visible on the
white background provided. On the other hand, any filter type will obscure some category of
plastic. The GF filters were compatible with Nile red and thus we were easily able to move
forward with Nile red staining and subsequent fluorescent microscopy.
The GF filters showed varying compatibility with µFT-IR and µRaman (discussed
below). However, for both µFT-IR and µRaman, it is useful to present the microplastic for
characterization on a filter paper/membrane that has a smooth surface, in terms of texture and 3D
character. Accumulating microplastic on smooth and flat filters improves the quality and
efficiency of µFT-IR and µRaman analyses, as well as the ability of confocal microscopes to
focus during visual examination and photo-documentation of samples. GF filters are comprised
of overlapping glass microfibers and present a rough and textured surface. Fragments can embed
in the microtopography of such 3D textured surfaces, impeding their characterization, and the
textured background of the filter itself can prevent the use of fragment-finder software that
depends on strong microplastic-filter contrast for reliable use. Oppositely, polycarbonate filters,
have a smooth uniform surface with little 3D character. Any filter’s texture can be easily checked
under a microscope at the magnification chosen for terminal analysis57 and we encourage novice
researchers to do so. Lastly, different sample processing steps can warp filters, sometimes
permanently, no longer permitting them to lie flat. Of greatest concern here is the “ridging”
which can occur when using vacuum filter funnels that have an elevated and textured platform.
We suggest researchers use traditional borosilicate glass vacuum filter platforms that are uniform
and flat.
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Working Solutions
We are just on the cusp of our wider scientific microplastic community more broadly
acknowledging that working solutions used in the sample processing steps can be a significant
source of secondary contamination32,36,58,59 in microplastic samples and, thus, all working
solutions must be pre-filtered using filter pore-sizes less than that detected by the specific study’s
terminal analysis. Surprisingly, this includes Milli-Q treated deionized (DI) water; water from
the Millipore system used in this study has on average 3.4 microplastics per L (Fig. 8a) and
requires double filtering prior to use with samples.

Figure 8. Representative photographs of different contaminants. Select fibers and fragments

filtered out of “raw” (i.e., not doubly filtered) (a) Milli-Q deionized water, (b) one cluster of
secondary contamination encountered on a Nile red stained filter, and (c) fluorescent (top) and
visual (bottom) pictures of a GF filters dyed with Nile red.

The Nile red used in this study was not pre-filtered, which was an oversight. It was made
up with high-performance liquid chromatography (HPLC)-grade acetone and n-hexane in DFDI
pre-rinsed glassware. This level of good laboratory practice did not suffice. One of the two Nile
red blank fibers visually examined under the Raman confocal microscope was clean, while the
other yielded a single patch of small blue fragments (Fig. 8a). Nile red is likely a source of
heterogenic secondary contamination that can easily be eliminated via pre-filtering in future
studies.32

Terminal Analyses
FT-IR Microscope. FT-IR microscopes are a powerful tool that can capture the polymeric signal
of microplastics, cellulose and other materials.38,39,60,61 These microscopes have several
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configurations to interface with samples: transmission mode (requiring samples placed within IR
transparent windows), ATR using various sized refractive crystals in direct contact with samples,
and reflection mode. In addition, several detector configurations are available with differences in
sensitivity, expense and speed: deuterated triglycine sulfate (DTGS), MCT, MCT-linear arrays,
and focal point array (FPA; multiple detector elements in a large array). For application to
microplastic research, transmission and µATR with a signal MCT detector (faster and higher
sensitivity compared to DTGS) are useful for larger fragments and fibers that can be readily
picked from environmental samples and placed in transmission cells or on a sturdy surface in
contact with a µATR crystal. Smaller fragments and fibers (<100 µm) that challenging to move
can be analyzed via µATR on their original collection surface.
Whole filters with areas from 95 to 1735 mm2 (i.e., circular filters with diameters of 11 to
47 mm) that are used to isolate fragments and fibers from environmental matrices require a
different approach. With environmental matrices, it is common that hundreds of fragments and
fibers are isolated on a filter and/or items <100 µm in size are abundant on the filter. Under such
conditions, it is not practical or physically possible to move each item on the filter into a
transmission cell for analysis. Further, it is time prohibitive to move item by item with a µATR
crystal cleaning in between each measurement. Indeed, there are various sized µATR crystals
available with contact areas in the 0.008 to 0.8 mm2 range (corresponding to 0.1 to 1 mm crystal
diameters) that can be coupled with FPA detectors and utilized in fast mapping modes. However,
to map a 95 mm2 filter with a 0.8 mm2 ATR would require >100 contact points on the filter, with
cleaning needed between each measurement and the risk that any given contacted item could be
removed and lost from the sample during crystal clearing. And, the heterogeneity of fragments
and fibers on a filter in terms of thickness and malleability make it difficult to establish a
consistent pressure of each item against a larger ATR crystal.
For filters, operating in reflection mode with an MCT linear array or FPA detector, which
can collect multiple spectra over a larger area at once, is more practical. For example, the
Thermo Fisher Nicolet iN10 MCT linear array comprises a 2 x 8 array of MCT elements and,
through automation, can map a 1.2 x 1.2 mm area in 4.5 minutes with a 25 x 25 µm spatial
resolution, 16 cm–1 spectral resolution, and 1 scan per location. In this mode, fully mapping an
11 mm filter will require roughly six hours and produce a considerably large spectral data set.
Looking forward, condensing or altering sample volumes samples to end with a smaller diameter
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filter and techniques for filter subsampling will be important to improve efficiency.22,62 Another
promising approach is reducing the area of a filter that is analyzed via fragment finder software,
which minimizes the amount of data generated per filter and improves spectral quality as specific
fragments are targeted rather than mapping the gross area of a filter. Challenges with reflection
mode include textured fragments or fibers with high refractive indices can interfere with IR
signals,39 and more significant, the signal from the filter material can interfere with the spectra of
the fragment or fiber given that many common filters have a strong IR signal (e.g., cellulose
acetate, glass microfiber). Gold coated filters provide the optimal substrate with minimal IR
signal and reflective properties that improve the fragment or fiber IR signal. Pricewise gold
filters may be cost prohibitive for many labs and other options, such as silica, aluminum oxide
and reusable gold coated slides are being investigated.13,62–65
Additional considerations with µFT-IR involve signal character, general interferences,
and detection limits. FT-IR is a bulk technique such that it is easier to characterize components at
percentage levels meaning, in plastics, the polymer itself will often give a stronger signal than
the trace components such as dyes and additives. The weaker signal from dyes and additive can
require further sophisticated sample preparation in compression cells followed by analysis in
transmission mode and possibly spectral subtraction or multicomponent spectral matches to
reference spectra in databases for detection. Certain materials, such as glass fibers, silicate
minerals, and water yield strong signals that can interfere with important polymeric fingerprint
regions. Further, FT-IR is considered resolution-restricted to larger fragments and fibers >10–15
µm due to limits in diffraction of the source light and physical contact between the ATR crystal
and sample.60,65 While ATR can push this diffraction limit with high refractive index crystals like
Germanium and collect a spectrum from objects as small as 1 µm (paired with a sensitive
detector), these are practical for point measurements on selected fragments or fibers. In this
study, interference from glass microfibers drove our use of a µATR coupled with a single MCT
detector on selected fragments and fibers removed from our GF filters to obtain clear and
identifiable spectra.

Raman Microscope
Raman complements FT-IR well in that it is an excellent tool for examining small fibers and
fragments (<20 µm) as well as characterizing dyes and additives that comprise a small portion of
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a material’s chemical makeup.38,39,60 Challenges associated with Raman include developing an

ideal sample presentation, minimizing fluorescence, and working in conjunction with signals
from dyes and additives that overlay the polymeric backbone (these signal issues are quickly
being addressed with new method development, technological advancements, and mathematical
corrections).41 Our experience with each of these aspects in this study are outlined below.
Common substrates used for sample presentation with µRaman at present include polycarbonate,
aluminum coated polycarbonate, aluminum oxide, silicon filters or glass slides, gold coated glass
slides, and glass petri dishes.40,57 Note that gold coated polycarbonate filters are only useful if
they are stretched flat. Any warping of the surface leads to strong contrast and interferes with
location of fragments on the filter surface when viewing through the confocal microscope. These
substrates work to minimize background noise and fluorescence, while maximizing Raman
signal and microplastic-filter contrast.57 In this study, the original GF filters from the sample
collection process were used for sample presentation on the µRaman. The use of the original
filters prevented any microplastic loss or secondary contamination that may occur during transfer
to a new filter material. Further, those glass microfibers on GF filters readily break and transfer
with the microplastic to the new filter material. The presence of transferred glass microfibers can
interfere with microplastic characterization. That said, our GF filters were reasonably compatible
with µRaman. The Raman signal from two different fibers (one from DI water and one from an
air sample) was tested under different conditions: (i) on a dry GF filter, (ii) on a wet GF filter,
and (iii) on a glass slide. We accomplished this by carefully moving the fibers with a pair of fine
tweezers from surface to surface. The glass slide yielded the strongest signal, followed by the
wet GF filter and then the dry GF filter (Fig. S1, Supplemental Material). Additionally, wetting
the GF filter allowed the glass microfibers to flatten, reducing their 3D nature, improving the
focus of the confocal microscope and reducing the glass microfiber interference with the Raman
signal from microplastics.
As mentioned in other studies, the interference of fluorescence with the Raman signal is a
general challenge in microplastic characterization.38,41,57 Raman signals can be 104 to 108 less
efficient than fluorescent signals,66 especially in strongly colored plastic or plastic containing
fluorescent dyes. This challenge can be met with several approaches that are described in Araujo
et al.41 These approaches involve the application of different laser wavelengths, photobleaching,
variation in sample presentation (e.g., aluminum polycarbonate filters)57 and algorithms to
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remove fluorescent signals from spectra.40 Unfortunately, fluorescent signals that overpowered
the Raman signal leading to highly elevated baselines were frequent in our sample set and
attempts at different laser wavelengths and/or intensities were only sometimes useful. This
decreased the overall number of successful spectra we were able to generate.
Plastics that are colored, but not fluorescent at the laser wavelength used for analysis, can
yield strong Raman spectra; however, the Raman signal from dye or additives in the plastics may
overpower the Raman signal of the polymeric material (e.g., Käppler et al.39). In this study,
multicomponent library searches of the KnowItAll database with Bio-Rad ExpertID software
were dominated by dyes and additives, with low to no signal from the polymeric backbones.
Subtracting the dye spectra from the original sample spectra yielded clearer polymer matches
only in some cases, indicating the interference from the dye signal was too strong to pull out the
polymer signal in many cases. Dyes and additives are associated with most manufactured
materials and (when associated with specific materials) provide relevant information for
characterization of fibers or fragments from challenging environmental matrices.39 For example,
dibutyl dichlorotin is a heat stabilizer associated with PVC that was frequently detected in our
samples (Table S1).
We evaluated a spectral range of 200–2000 cm–1, similar to several previous studies.22,37
However, for future studies, moving to a range of 400–3200 cm–1 may be more effective at
capturing the microplastic polymeric backbone.23,36,39,40 There is a cluster of Raman peaks in the
2780–2980 cm–1 range resulting from CH–CH–CH groups stretching vibrations and are strongly
3

exhibited by many of our more common plastics (Figure 4 in Käppler et al.39). The obvious
downside here is that such a wider spectral window can double to triple analysis time as the full
400–3200 cm–1 range cannot be captured in a single detector window. Two to three windows
would need to be analyzed and coadded (depending on the excitation laser used). This additional
analysis time would need to be evaluated and weighed against the quality of spectra produced
and their ability to identify polymeric signals.

Nile Red Staining

Aluminum oxide membrane and polycarbonate filters are commonly used with Nile red for
sample presentation,32–34 however, the original GF sample filters used in this study were easily
adapted to Nile red. The glass microfibers are compatible with the n-hexane and acetone solvents
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within the Nile red staining solution and other than some secondary contamination from the Nile
red solution (Fig. 8), do not appear to have any significant fluorescent interference under the
conditions utilized in the study (Fig 8). A check of the planned sample presentation is important
because fluorescent interference can occur and add to background noise.34 During traditional
microscopy of GF sample filters, yellow organic-like material (often irregularly elongated along
one axis and “lumpy”) and degraded carbonaceous material (black carbon) were observed. This
black carbon had no fluorescent signal, while the yellow organic-like matter had an infrequent,
but variable fluorescent signal suggesting possible algal or other microbial origins. These
observations, along with the high abundance of cellulose shown in the FT-IR data, indicate a
digestion step to reduce the presence of organic material prior to Nile red staining is necessary
for suspended air samples. As with all of our other microplastic work, a digestion step for
airborne sampling will likely improve the overall accuracy of visual and Nile red counts, given
that our original secondary contamination can be minimized. Overall, Nile red quantification that
includes a sample digestion step and is initially validated with FT-IR or Raman characterization
and matrix spikes,32 can provide an approachable method for evaluating microplastic
contamination densities in environmental samples.

Conclusion
This study presents some of the first data on microplastic densities in air, specifically comparing
indoor and outdoor environments. Suspended fibers and fragments were filtered from air for
subsequent microplastic analysis in paired, indoor and outdoor locations around the CSUCI
campus in coastal California. Sampled fibers and fragments were assessed for microplastic
content using four microscopy techniques that are presently being evaluated by the larger
scientific community for their effectiveness in the field of environmental microplastic
contamination. Each technique was able to provide different information about the source,
pathway, and/or composition of our sampled microplastic. Traditional microscopy, the most
broadly used technique, was useful for gross counts of microplastic density, investigations into
the different microplastic morphologies present, and especially with observations of spatial
trends across the university campus. We found elevated fiber densities in indoor areas with high
foot traffic, potentially from microfibers shedding off synthetic clothing. Fluorescent microscopy
with a pre-digestion and Nile red staining, as well as prior method validation, can improve the
DOI: 10.1177/0003702820920652

accuracy of traditional microscopy. In our case, Nile red counts picked up a large population of
small (<50 µm) indoor air fragments that would otherwise not have been visualized or counted.
Because no digestion was carried out in the study, we cannot rule out the biogenic origin of these
small fragments as opposed to a polymeric origin, but their presence is an interesting observation
that stemmed from Nile red staining.
The two spectrographic techniques we employed, µFT-IR and µRaman, showed PE and
PET fibers concentrated indoors where people and furniture with synthetic PE and PET clothing
are also concentrated and air is minimally diluted with fresher/cleaner air masses. Outside, we
observed an abundance of PVC fibers common to exterior fabric and canvas. These chemical
fingerprinting techniques are, as present, only semi-automated and require a certain skill set to
produce high quality spectra and subsequent fingerprints. Thus, a number of recommendations
on sample preparation/presentation, instrument setup and technique strengths and weaknesses are
included herein as a resource to novice users.
Airborne microplastic represents a potential direct pathway for human plastic exposure
and an underappreciated source of microplastic transport into distant terrestrial and aquatic
ecosystems. As such this and similar studies highlight the need for comparable, robust global
data on the varying densities of microplastics in air as well as improved estimates of human
exposure, acute and chronic toxicities related to respired microplastics, and potential
mechanisms and policies for future source reductions. The multiple assessment techniques
applied herein will each be important in providing a more coherent picture of microplastic
contamination in air as our nascent field of environmental microplastic characterization works
towards these larger goals.
Acknowledgments
The authors would like to thank Ralph Diego, recent graduate of California State University
Channel Islands (CSUCI) for his non-stop collection of indoor and outdoor air samples; Eunah
Lee, Horiba Raman Project Manager at HORIBA Scientific, for training, mentoring, and use of
the µRaman instrument; Andrew Whitley, VP Sales and Business Development at HORIBA
Scientific for providing us with this opportunity; CSUCI Students: Julie McHorney, Emmie
Wordin, Nathan Shaprio, and Melvin Kim for helping in various tasks as needed to complete the
data analysis, and lastly the Cause and Tracy S. Hanna Scholarship for supporting Ralph Diego’s
DOI: 10.1177/0003702820920652

data collection.

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DOI: 10.1177/0003702820920652

Tables

Table I. Air sampling locations on the California State University Channel Islands campus
(Camarillo, California). Note: for simplicity, the “pair” descriptor will be used in
subsequent data figures.
Pair Indoor sites Outdoor sites

Location Descriptor Replicates Location Descriptor Replicates

Modoc Modoc Laboratory 2 Facilities Parking lot 3

Sierra Sierra Foyer 2 Mall Promenade 2

Broome Broome Library 3 Fountain Reflecting pool 2

ESRM ESRM Laboratory 3 Patio Terrace 2

Union – – – Union Plaza 2

Table II. Size of airborne microplastic fibers and fragments at CSUCI. Note: these
measurements exclude items smaller than 20 μm.
DOI: 10.1177/0003702820920652

Fiber length (µm) Fragment size (µm)

n Min Max Mean SD n Min Max Mean SD

Indoor 239 22 8961 641 810 316 20 850 58 55

Outdoor 63 25 2061 616 536 50 51 408 104 64

Table III. Summary of the mean and standard deviation (SD) of all sample GF filters
analyzed in each category and the polymeric content of the fragment and fiber subset
analyzed by µFT-IR and µRaman. Note the raw and blank (i.e., procedural control)
corrected values for visual and Nile red counts are included. Were we to have conducted a
digestion of filters, the cellulose signals would have disappeared, so we present these for
ease of comparison with other publications.

Indoor per m 3
Outdoor per m 3

Fiber Fiber
(mean ± SD) Fragment (mean ± SD) (mean ± SD) Fragment (mean ± SD)

Visual quantification

Raw 11.4 ± 7.8 7.6 ± 4.8 2.6 ± 1.4 16.5 ± 6.0

Corrected 9.8 ± 7.3 6.8 ± 5.2 1.5 ± 1.1 15.5 ± 6.1

Nile red
quantification

Raw 3.8 ± 2.9 14.6 ± 8.5 1.0 ± 0.8 6.6 ± 3.3

Corrected 3.3 ± 2.9 12.6 ± 8.0 0.6 ± 0.6 5.6 ± 3.2

Spectrographic characterization
DOI: 10.1177/0003702820920652

Indoor Air Fiber + Fragment Polymeric Content Outdoor Air Fiber + Fragment Polymeric Content
(%) (%)

FT-IR 15 4.8

25 excluding cellulose 13 excluding cellulose

Raman 63 47

Supplemental Material
Microplastics Differ Between Indoor and Outdoor Air Masses:
Insights From Multiple Microscopy Methodologies
Emily Gaston,1,* Mary Woo,1 Clare Steele,1 Suja Sukumaran,2 Sean Anderson3
1
California State University Channel Islands, Environmental Science and Resource Management
Program, CA 93012 USA
2
Thermo Fisher Scientific, San Jose, CA 95134 USA
3
California State University Channel Islands, Environmental Science and Resource Management
Program, PIRatE Lab, CA, 93012 USA
*Corresponding author email: Emily.Gaston@csuci.edu
DOI: 10.1177/0003702820920652

Figure S1. Spectra of the same fiber on a glass slide, dry GF filter, and wet GF filter.

Table S2. List of chemical types, counts, and application identified as polymeric by
µRaman.
Counts (items) Percent Application

Chemical Type

di-n-butyldichlorotin 15 42% heat stabilizer in PVC

Valfor 100 Zeolite 4 11% heat stabilizer in PVC

polyphosphoric acid 4 11% additive to polymeric asphalt

1,2,3 triazole 4 11% photostablizer in polymers

copper phthalocyanine 1 3% common dye in polymers

PVC 2 6% widespread consumer use in plastic

PE 2 6% widespread consumer use in plastic

Resin 2 6% widespread consumer use in plastic

PS 1 3% widespread consumer use in plastic

ABS 1 3% widespread consumer use in plastic

acrylic 1 3% widespread consumer use in plastic

PC 1 3% widespread consumer use in plastic

Table S1. DI water and microplastic counts.

DOI: 10.1177/0003702820920652

Ora Yell Gre Blu Pur Bro Bla Tot Tota

Red nge ow en e ple wn ck al l Fiber
Wa Repli Volu Fib Fibe Fibe Fib Fib Fib Fibe Fib Fil Fragm fib plast richn
ter cate Date me ers rs rs ers ers ers rs ers m ents ers ics ess

DI
Wa 3/13/
ter 1 19 1 0 0 0 0 2 1 0 0 2 0 5 5 2

DI
Wa 3/13/
ter 2 19 1 0 0 0 0 2 4 0 0 1 0 7 7 2

DI
Wa 3/13/
ter 3 19 1 0 0 0 0 1 1 0 0 1 0 3 3 2

DI
Wa 3/13/
ter 4 19 1 0 0 0 1 0 0 0 0 0 0 1 1 1

DI
Wa 3/13/
ter 5 19 1 0 0 0 0 3 1 0 0 1 0 5 5 2

DI
Wa 3/13/
ter 6 19 1 0 0 0 0 0 1 0 0 0 0 1 1 1

DI
Wa 3/13/
ter 7 19 1 0 0 0 0 1 0 0 0 1 0 2 2 1

DI
Wa 3/15/
ter 8 19 1 0 0 0 0 4 2 0 0 0 0 6 6 2
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DI
Wa 3/15/
ter 9 19 1 1 0 0 0 3 1 0 0 0 2 5 7 3

DI
Wa 3/15/
ter 10 19 1 0 0 0 0 0 0 0 0 0 1 0 1 0

DI
Wa 3/15/
ter 11 19 1 0 0 0 0 2 1 0 0 0 0 3 3 2

DI
Wa 3/15/
ter 12 19 1 1 0 0 0 1 1 0 0 0 0 3 3 3

DI
Wa 3/15/
ter 13 19 1 0 0 0 0 1 2 0 0 1 0 4 4 2

DI
Wa 3/15/
ter 14 19 1 0 0 0 0 0 0 0 0 0 0 0 0 0

DI
Wa 3/15/
ter 15 19 1 0 0 0 0 0 3 0 0 0 0 3 3 1

DI
Wa 3/15/
ter 16 19 1 0 0 0 0 0 0 0 0 2 0 2 2 0

DI
Wa 3/26/
ter 17 19 1 0 0 2 0 3 6 0 0 1 0 12 12 3
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DI
Wa 3/26/
ter 18 19 1 0 0 0 0 2 3 0 0 1 0 6 6 2

DI
Wa 3/26/
ter 19 19 1 0 0 0 0 0 3 0 0 1 0 4 4 1

DI
Wa 3/26/
ter 20 19 1 0 0 0 0 2 2 0 0 1 0 5 5 2

DI
Wa 3/26/
ter 21 19 1 0 0 1 0 2 2 0 0 3 0 8 8 3

DI
Wa 3/26/
ter 22 19 1 0 0 0 0 0 1 0 0 2 0 3 3 1

DI
Wa 3/26/
ter 23 19 1 0 0 0 0 1 0 0 0 1 0 2 2 1

DI
Wa 3/26/
ter 24 19 1 0 0 0 0 2 0 0 0 0 0 2 2 1

DI
Wa 3/26/
ter 25 19 1 0 0 0 0 0 0 0 0 0 0 0 0 0

DI
Wa 3/26/
ter 26 19 1 0 0 0 0 2 1 0 0 0 0 3 3 2
DOI: 10.1177/0003702820920652

DI
Wa 3/26/
ter 27 19 1 0 0 0 0 1 1 0 0 1 0 3 3 2

DI
Wa 3/26/
ter 28 19 1 0 0 0 0 3 0 0 0 0 0 3 3 1

DI
Wa 3/27/
ter 29 19 1 0 0 0 0 1 0 0 0 1 1 2 3 1

DI
Wa 3/27/
ter 30 19 1 0 0 0 0 0 1 0 0 2 0 3 3 1

DI
Wa 3/27/
ter 31 19 1 0 0 0 0 2 1 0 0 1 0 4 4 2

DI
Wa 3/27/
ter 32 19 1 0 0 0 0 1 0 0 0 0 0 1 1 1

DI
Wa 3/27/
ter 33 19 1 0 0 0 0 4 0 0 0 0 0 4 4 1

DI
Wa 3/27/
ter 34 19 1 0 0 0 0 1 1 0 0 0 0 2 2 2

DI
Wa 3/27/
ter 35 19 1 0 0 0 0 1 3 0 0 0 0 4 4 2
DOI: 10.1177/0003702820920652

DI
Wa 3/27/
ter 36 19 1 0 0 0 0 1 3 0 0 0 0 4 4 2

DI
Wa 3/27/
ter 37 19 1 0 0 0 0 1 0 0 0 0 0 1 1 1

DI
Wa 3/27/
ter 38 19 1 0 0 0 0 0 1 0 0 0 0 1 1 1

DI
Wa 3/27/
ter 39 19 1 0 0 0 0 0 1 0 0 0 0 1 1 1

DI
Wa 3/27/
ter 40 19 1 0 0 0 0 1 0 0 0 0 1 1 2 1

DI
Wa
ter Total: 2 0 3 1 51 48 0 0 24 5 129 134